Project Summary Co-delivery of Cas9 mRNA and targeted sgRNA is a promising strategy to achieve CRISPR/Cas gene editing in vivo with improved safety. Although great advances have been made in the delivery of short RNAs (siRNA, miRNA), the ideal chemical and formulation composition is largely unknown for longer RNA cargo (mRNA, sgRNA). We recently overcame this delivery challenge in reporting the first successful NP co-delivery of mRNA and sgRNA in vivo. In this grant proposal, we aim to fundamentally understand why zwitterionic amino lipid (ZAL) nanoparticles (ZNPs) are uniquely suitable and particularly efficacious for delivery of long RNAs. We hypothesize that the molecular balance of zwitterionic and cationic groups within NPs is essential for delivery of long nucleic acids, especially the noncovalent bonding forces at the interface between carrier molecules and RNAs. This hypothesis is supported by computational modeling that indicates the role of phospholipids is to solubilize RNAs inside of aqueous pockets within multi-component NPs. Indeed, we found experimentally that combining the chemical and structural roles of zwitterionic lipids and cationic lipids into a single lipid compound (ZAL) greatly improved delivery of long RNAs. In this proposal, we will develop (and fundamentally understand) improved delivery carriers for co-delivery of Cas9 mRNA and targeted sgRNA to enable safe and efficacious CRISPR/Cas gene editing in vivo. Completion of the proposed studies will: (1) Determine the functional roles of novel ZAL headgroups, linkers, and hydrophobic domain tails for co-delivery of mRNA and sgRNA; (2) Identify the physiochemical properties of efficacious long RNA (mRNA, sgRNA) lipid carriers that are correlated to the mechanism of intracellular delivery; and (3) Determine how the chemical structure of ZALs mediate cell and tissue specific CRISPR/Cas gene editing in vivo by utilizing a genetically engineered Lox-Stop-Lox tdTomato mouse that can reveal editing in any organ or cell. Cumulatively, this will open new avenues for CRISPR/Cas- based correction of genetic diseases by developing efficacious, safe, and clinically translatable nanoparticle carriers. Defining the specific interactions is a critical goal that would greatly improve delivery of long RNAs by numerous existing and future carriers. This broader impact may greatly accelerate the clinical development of mRNA and CRISPR/Cas therapeutics.